RNA-protein complexes are essential contributors to many important biological processes, including gene expression. An understanding of how proteins recognize RNA with high affinity and specificity will be valuable for describing and controlling these processes. The goal of this research program is to characterize the energetic contributors to RNA-protein complex formation. The proposed research will focus on the recognition of RNA by the RNA recognition motif (RRM), which is one of the most common RNA-binding domains. In the previous funding period, highly conserved aromatic amino acids that are involved in stacking interactions with RNA bases were found to be exceptional contributors to the stability of the U1A-RNA complex. In the current funding period, the molecular origins of the stabilization provided by these conserved aromatic amino acids will be investigated. Their contributions to stacking interactions in the complex, to the structures of the free protein and the complex, and to the conformational changes required upon binding will be probed by combining protein mutagenesis and synthetic modification of the RNA with structural studies, binding thermodynamic and kinetic measurements, and molecular dynamics simulations. To probe the generality of the results obtained with U1A, similar investigations with other RRM-RNA complexes will be performed. The comparison of binding in different RRM-RNA complexes will enable the identification of the energetic contributions that are held in common in RRM-RNA complexes. Finally, the RRM will be used as a template to develop small beta-hairpin peptides that bind RNA using phage display techniques. In particular, the ability of aromatic amino acids to stabilize beta-hairpin-RNA interactions will be investigated. Together, the proposed research will uncover new models for how aromatic amino acids control RNA-protein interactions. These models should be useful in the development of tools for the selective control of biological processes involving RNA-protein complexes.